Microparticulated vaccines for the oral or nasal vaccination and boostering of animals including fish

ABSTRACT

The invention relates to a composition and a method for manufacturing semi-dry or dry particles containing a mucoadhesive polymer and a bioactive agent such as, but not limited to, an Immunogenic Substance (e.g., a vaccine), that allows the oral or nasal administration and delivery of the bioactive agent essentially unaltered to mucosal surfaces in the animal, including an aquatic animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/260,661 filed Nov. 2, 2011, which is a U.S. national stageapplication of International Patent Application No. PCT/US2010/028767,filed Mar. 26, 2010, which claims priority to U.S. ProvisionalApplication No. 61/163,910 filed Mar. 27, 2009 and U.S. ProvisionalApplication No. 61/294,672 filed Jan. 13, 2010, the contents of whichare hereby incorporated in their entireties for all purposes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to encapsulated vaccines andmethods of making same, and more particularly, to oral vaccines thatrelease an embedded bioactive agent at the site of action along theforegut and hindgut of an animal and the vaccine is embedded in across-linked matrix that is protected by a glassy matrix of sugars.

2. Related Background Art

Many therapeutic agents, particularly vaccines, are mostly deliveredthrough the injectable route, which is traumatic, inconvenient,expensive, and may fail to induce an appropriate immunogenic response inthe mucosal tissues of the animal. In fact, most infections begin at themucosal surfaces, so immunization against these infective agents dependson the successful induction of a mucosal immune response. Whileparenteral vaccination is effective at eliciting a systemic immunity,oral vaccines can elicit mucosal immunity and also induce systemicimmunity by induction of circulatory antibodies. Oral vaccines are alsoeasier to administer and are less expensive to manufacture thanconventional vaccines. However, orally delivered bacterins or subunitvaccines have not been proven to be efficacious since the antigens aregenerally digested or modified by the stomach prior to presentation tothe immuno-responsive cells of the gut mucosa. It is recognized that onpassage through the stomach, the vaccine antigenic component(s) can berapidly inactivated by the gastric pH and digestive enzymes, and thuseffective systemic assimilation is compromised. A number of approacheshave been tested to provide an oral delivery vehicle that would transitthe stomach, but most have been unsuccessful at the commercial scale.

Polymer microspheres and lamellar particles (e.g., liposomes) can beemployed for mucosal administration of antigens. Because vaccinesthemselves may not be efficiently recognized and taken up by mucosallymphocytes, they typically need to be co-administered with penetrationenhancers or adjuvants. Different classes of polymer mixtures are knownfor potential use as mucoadhesives (Malik, Baboota et al. 2007). Theseinclude synthetic polymers such as poly (acrylic acid) (PAA),hydroxypropyl methylcellulose and poly-(methylacrylate) derivatives, aswell as naturally occurring polymers such as hyaluronic acid andchitosan. Chitosan has been extensively used for a variety ofapplications as a biomaterial for tissue engineering, wound healing, andas an excipient for drug delivery (Chopra, Mandi et al. 2006; Dang andLeong 2006). Chitosan has occasionally been tested as an adjuvant formucosal application (Kim, Kim et al. 2007), but it is typically applieddirectly to a mucosal surface such as intranasal application in order toobtain IgA response in the nasopharyngeal mucosa of terrestrial animals(Kang, Jiang et al. 2007). Chitosan has also been shown to possessuseful properties such as non-toxicity, high biocompatibility andnon-antigenicity.

Chitosan can be obtained through the deacetylation of chitin, the majorcompound of exoskeletons in crustaceans. Chitosan[a-(1˜4)-2-amino-2-deoxy-β-D-glucan], a mucopolysaccharide closelyrelated to cellulose, exhibits chemical properties that are determinedby the molecular weight, degree of deacetylation, and viscosity.Chitosan can form microparticles and nanoparticles that can encapsulatelarge amounts of antigens (van der Lubben, Verhoef et al. 2001; Davis2006). In the acidic environment of the stomach, chitosan retains itspositive charges that hold the particle together. It has been shown thatovalbumin loaded chitosan microparticles can be taken up by the Peyer'sPatches of the gut associated lymphoid tissue of higher vertebrates.Additionally, after co-administering chitosan with antigens in nasalvaccination studies, a strong enhancement of both mucosal and systemicimmune responses in mice was observed (van der Lubben, Verhoef et al.2001).

As a result of its interesting properties, chitosan has become thesubject of numerous scientific reports and patents on the preparation ofmicrospheres and microcapsules. Chitin and chitosan are beingextensively used in the pharmaceutical industry (cosmetics, contactlenses, artificial skin, wound dressing), paper making, photography,solid state batteries, waste water treatment, chromatography, dietarysupplements and animal feed. Processing techniques for the preparationof chitosan microspheres have been extensively developed since the1980s. Several processing approaches have been proposed includingionotropic gelation with an oppositely charged, simple or complexcoacervation, emulsification/solvent evaporation and, more recently,spray drying (Huang et al. 2003). Chitosan microspheres obtained byspray drying are characterized by high sphericity and specific surfacearea, which are important parameters for application in thepharmaceutical field (Rege, 2003).

One particular advantage of chitosan is its ability to form a gel matrixwith counter-ions such as sodium tripolyphosphate (TPP) (Bodmeier et al.1989, Shiraishi et al. 1993, Calvo et al. 1997). TPP is a non-toxic andmultivalent anion. It can form either intermolecular or intramolecularlinks between positively charged amino groups of chitosan and negativelycharged counter-ion of TPP (Aral and Akbuga 1998; Shu and Zhu 2000).

Against this background, there is a need for an attractive compositionand manufacturing method for an oral delivery system that is costeffective, simple to prepare, and also permits prolonged storagestability while maintaining a high loading capacity for the bioactiveagent with retention of its in-vivo immunogenicity. Further desirablebenefits of the delivery system would include the accurate dosing ofbioactive agent, and the ability of stabilizing and protecting thebioactive agent during the manufacturing process itself (e.g. pelletingor extrusion of a food of feed product). It is the objective of thepresent invention to provide a composition and a manufacturing method tomeet these needs.

SUMMARY OF THE INVENTION

The present invention provides a composition and a method for themanufacturing of a mucoadhesive delivery system for the oral vaccinationof animals. The mucoadhesive delivery vehicle releases the vaccine atthe site of action (i.e., the Gut Associated Lymphoid Tissue; GALT)along the foregut and hindgut of the animal. In a preferred embodimentof the invention the mucoadhesive delivery vehicle is incorporated inthe regular food or beverage normally consumed by the animal.

The mucoadhesive delivery vehicle is provided in a form of dry orsemi-dry particles comprising an immunogenic substance (i.e., thevaccine), which is embedded in a composite matrix of cross-linkedmucoadhesive polymer and protected by sugars.

It is an object of the invention to provide a manufacturing process of amucoadhesive delivery vehicle for vaccination of animals and, in oneembodiment, to aquatic animals.

Remarkably, the present inventors have found a way to produce largequantities of mucoadhesive particles containing vaccines with minimaldrying efforts under sterile conditions and while using only food gradeingredients. In addition, when incorporated in a food product andadministrated orally, the particles elicited both mucosal and systemicimmune responses.

Thus, according to the present invention, there is provided acomposition and a method of preparing a particle comprising a bioactiveagent that is embedded in one or more mucoadhesive polymers, the one ormore mucoadhesive polymer being further embedded in a glassy matrix,wherein the glassy matrix comprises: 10-60 wt % of sugars, 3-10 wt % ofa oligosaccharides and 1-10 wt % of electrolytes, wherein theelectrolytes, such as di- or poly-valent anion or cation compounds, actas cross-linking agents.

The present invention also provides a process of preparing a particleencapsulating a bioactive agent, the method comprising the steps ofdissolving one or more mucoadhesive polymers in aqueous solution,admixing the bioactive agent under ambient temperature and slightlyacidic conditions, extruding the slurry into a counter ion solution toform firm hydrogel beads or strings and saturating the hydrogelparticles with sugars.

In additional embodiment, the sugar saturated hydrogel particles arefurther dehydrated by desiccation to reduce the moisture content tobelow 20%.

In one aspect the semi-dry particle materials are subjected to furtherdrying and milling to obtain microparticulated powder containing thebioactive agent and having moisture content below 10%.

In another aspect, the present invention provides a semi-wet particle asdescribed above, where the hydrogel particles are chopped for furthersize reduction without further drying.

In yet another aspect, the present invention provides a bioactive agentembedded in a mucoadhesive polymeric matrix and is further stabilizedand thermo-protected by glassy matrix of sugars.

In still another aspect, the present invention provides a process ofpreparing a particle containing a bioactive agent where the hydrogelparticles are saturated with sugars which significantly reduces theamount of free water in the particles and the associated need forextensive drying step.

In still another aspect, the present invention provides a process ofpreparing a particle containing a bioactive agent where all ingredientsincluding salts and solutions are food grade ingredients, non-toxic,biodegradable and naturally occurring ingredients.

Yet another aspect of the present invention provides an immunogenicparticle comprising a bioactive agent, one or more mucoadhesive polymerand an emulsifier all of which are cross-linked with a phosphatecontaining agent to form a hydrogel particle, and wherein the hydrogelparticle is embedded in a sugar matrix.

The present invention has a number of unexpected advantages over theprior art. Thanks to the specific formulation and process conditions,the water content in the particles is very minimal while at the sametime the formula also stabilizes the sensitive bioactive. A furtherimportant advantage is the excellent thermo-stability of theencapsulated ingredient. Without being bound by theory, it is believedthat the amorphous sugar glassy structure created around the bioactiveagent provides protection against the deleterious effects of heat andoxidation. This permits the particles of the present invention to beincorporated in a food product, the preparation of which entailspressure, sheer force, and heat treatments. Further advantages includethe free-flowing characteristics of the particles of the invention aswell as ability to control the particle size over a wide range from 5microns to over 5000 micron.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic side view of the apparatus for producing theparticles of the current invention.

FIG. 2 shows the hydrogel strings before (upper picture) and after(bottom picture) harvesting from vessel B.

FIG. 3 shows the optimum treatment conditions for immunogen release fromthe particles.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

Unless otherwise defined herein, scientific and technical terminologiesemployed in the present disclosure shall have the meanings that arecommonly understood and used by one of ordinary skill in the art. Also,as used herein and in the claims, the terms “at least one” and “one ormore” have the same meaning and include one, two, three or more.

In describing the present invention, the following terminology is usedin accordance with the definitions set out below.

“Bioactive Agent” refers to naturally occurring, synthetic, orsemi-synthetic materials (e.g., compounds, fermentates, extracts,cellular structures) capable of eliciting, directly or indirectly, oneor more physical, chemical, and/or biological effects. The bioactiveagent may be capable of preventing, alleviating, treating, and/or curingabnormal and/or pathological conditions of a living body, such as bydestroying a parasitic organism, or by limiting the effect of a diseaseor abnormality. Depending on the effect and/or its application, thebioactive agent may further be referred to as a pharmaceutical agent(such as an immunogen, a prophylactic agent, a therapeutic agent), adiagnostic agent, and/or a cosmetic agent, and includes, withoutlimitation, vaccines, prodrugs, affinity molecules, synthetic organicmolecules, polymers, low molecular weight molecules, proteinaceouscompounds, peptides, vitamins, steroids, steroid analogs, lipids,nucleic acids, carbohydrates, precursors thereof, and derivativesthereof. The bioactive agent may also be a nutritional supplement.Non-limiting nutritional supplements include proteins, carbohydrates,water-soluble vitamins (e.g., vitamin C, B-complex vitamins, and thelike), fat-soluble vitamins (e.g., vitamins A, D, E, K, and the like),and herbal extracts. The active agent may be commercially availableand/or prepared by known techniques.

“Microencapsulation” is defined as a process that produces a compositioncontaining a bioactive agent that is in the form of a microparticle inthe size range of 10 to 5000 μm, or a composition that can be milled toa microparticle in the size range of 10 to 5000 μm.

“Complex” is defined as interaction between two molecules or portions ofthe same molecule through noncovalent interactions such as coordinationbonds, electrostatic interactions, hydrogen bonding interactions, andhydrophobic interactions.

“Particle” refers to a particulate that is solid (includingsubstantially solid or semi-solid, but excluding gel, liquid and gas),having an average geometric particle size (sometimes referred to asdiameter) of less than 5 mm, preferably 500 microns or less, morepreferably between 100 microns and 5 microns. Particles may be formedfrom, in part or in whole, with one or more non-limiting materials, suchas the bioactive agents, mucoadhesive polymers, carriers, polymers,stabilizing agents, and/or complexing agents disclosed herein.

An “Immunogen” or an “Immunogenic Substance” is defined as a bioactive,a substance or a composition of matter, which is capable of mounting aspecific immune response in an animal. Immunogenic substances wouldinclude immunogenic peptides and proteins including mixtures comprisingimmunogenic peptides and/or proteins (e.g., bacterins); intact inactive,attenuated, and infectious viral particles; intact killed, attenuated,and infectious prokaryotes; intact killed, attenuated, and infectiousprotozoans including any life cycle stage thereof, and intact killed,attenuated, and infectious multicellular pathogens, recombinant subunitvaccines, and recombinant vectors to deliver and express genes encodingImmunogenic proteins (e.g., DNA vaccines).

“Vaccination” is defined as a process that results in a specific immuneresponse generated by an animal against an immunogen or an immunogenicsubstance.

A “Mucoadhesive Delivery System” or “Mucodhesive Delivery Vehicle” isdefined as a composition that results in the delivery of bioactiveagent, an immunogen or an immunogenic substance to the desired locationin the intestinal or nasal mucosa.

“Mucoadhesive Polymer” refers to a natural, synthetic, or semi-syntheticmolecule having two or more repeating monomer units in a main chain orring structure. Polymers broadly include dimers, trimers, tetramers,oligomers, higher molecular weight polymers, substituted derivativesthereof, and mixtures thereof. The polymer may be ionic or non-ionic,may be neutral, positively charged, negatively charged, or zwitterionic,and may be used singly or in combination of two or more thereof.

A “Mucoadhesive” molecule is a component of a mucoadhesive deliverysystem that specifically binds to mucosal tissues. Such moleculesinclude, but are not limited to chitosan, alginate, hyloronic acid andcationic guar.

“Amorphous” refers to the glassy state of materials and constructionsthat lacking crystallinity or otherwise non-crystalline.

A “Glassy Matrix” for the purpose of the present invention, is anamorphous solid characterized by viscosities and an extremely lowmolecular mobility. The presence of a glassy state can be confirmed byestablishing characteristic differential scanning calorimetry curves,for which particles are generally brought to the rubbery state by slowand continuous heating of the material.

“Semi-Dry or Semi-Wet” refers to a state that includes at leastsubstantially solid and/or semi-solid, but excludes gel, liquid, andgas.

“Cross-Link,” “Cross-Linked” and “Cross-Linking” generally refer to thelinking of two or more materials and/or substances, including any ofthose disclosed herein, through one or more covalent and/or non-covalent(e.g., ionic) associations. Cross-linking may be effected naturally(e.g., disulfide bonds of cystine residues) or through synthetic orsemi-synthetic routes. As described herein, cross-linking refer toconverting polymeric slurry into a firm structure of hydrogel particlesusing salts having counter ions.

Oral administration of vaccines offers several advantages. Dosages canbe administered to a large number of animals via the food or water withminimal labor and stress to the animal. Adverse immune reactionsfollowing oral administration are also much less likely to occur. Formeat producing animals, oral administration has the additional advantageof avoiding common reactions or infections at injection site, brokenneedles, or the use of highly reactive adjuvant. These reactionsdecrease the value of the animal at harvest.

An effective oral delivery system requires a delicate balance amongfactors such as the simplicity of preparation, cost effectiveness, highloading level of the bioactive agent, controlled release ability,storage stability, and effective immunogenicity of the components. Themethod and process described herein offers significant advantagescompared to other particulate delivery systems, including theconventional micro- and nano-encapsulation systems. It is also expectedthat the problems of instability, low loading level, and costeffectiveness are better resolved with the polymeric mucoadhesive systemof the current invention.

The present invention provides a composition and a method formanufacturing of mucoadhesive particles containing one or more bioactiveagents. Although various bioactive agents may be microencapsulated inaccordance with this invention, the invention will be described belowprimarily with reference to the microencapsulation of ImmunogenicSubstances that are bacterin vaccines. Thus, according to one preferredembodiment, the present invention enables the oral delivery of animmunogenic vaccine useful in the prevention of disease in animalsincluding aquatic animals.

Preparation of Bioadhesive Polymer slurry: A mucoadhesive polymer, suchas but not limited to chitosan, at a concentration of 1-10% (w/w), isdispersed in 1-5 N acetic acid solution at a temperature in the range of20° C. to 65° C. until fully dissolved. Indigestible short chainoligosaccharide components may be added to improve protection of theantigen from stomach acidity, bile acids, and proteases, and to increasethe intestinal adsorption of the bioactive agent. Examples of materialsthat could be used include, but are not limited to, inulin andfructooligosaccharides (FOS). The pH of the slurry is then brought toabout 5.8 and 0.5-10% (w/w) of a natural emulsifier such as lecithin isadded to form a stable emulsion. One or more mono or disaccharides arethen added to achieve a saturated emulsion with a concentration of from5-50% (w/w). Without being bound by theory, it is believed that theemulsion is stabilized by the interaction between positive charge of themucoadhesive polysaccharide, the emulsifier, and hydroxyl groups of thesugars and oligosaccharides. The increased hydrophobicity and elasticityof the mucoadhesive polysaccharide and emulsifier helps delay or preventpenetration of water or gastric juices into the matrix once formed intoparticles. In addition, the sugars and phospholipid complex increasesthe stability of the bioactive agent and protects the agent againstheat. At the same time, the added sugars dramatically reduce the freewater in the slurry, allowing for faster dehydration and drying. The pHof the slurry is then gradually increased to about pH 6.2 by theaddition of base and a solution containing the bioactive agent added.

Hydrogel formation. Cross-linking is used to promote the formation ofstable hydrogel particles. Various cross-linking agents have been usedin fixing polymeric gels. These cross-linking agents are mostlysynthetic chemicals such as formaldehyde, glutaraldehyde, dialdehyde,starch, glyceraldehydes, cyanamide, diimides, dimethyl adipimidate,diisocyanates, and epoxy compounds. However, these chemicals are allhighly cytotoxic which will restrict their utility in food applications.Of these, glutaraldehyde is known to have allergenic properties and iscytotoxic at concentrations greater than 10-25 ppm and as low as 3 ppmin tissue culture. It is the purpose of this invention to provide across-linking agent suitable for use in food applications that is withinacceptable cytotoxicity and that forms stable and biocompatiblecross-linked products. To achieve this goal, a food grade product havinga Generally Recognized As Safe (GRAS) status as a cross-linking agent(sodium triphosphate) has been used to form stable hydrogel particles.The resulting solution/suspension is then dropped or extruded into across-linking solution containing water-soluble phosphate salts. Uponcontact, a salt exchange reaction (cross-linking) takes place, resultingin the formation of hydrogel beads or linear threads in which thebioactive agent is retained. The resulting suspension of particlescontaining the embedded bioactive agent is then soaked in a sugarsaturated solution, collected, and dewatered to form semi-dry particlesthat can be further dried by a number of means well known in the artsuch as freeze drying, vacuum drying, spray drying, and the like, andmilled to produce fine powder having a size range of from 5 to 5000microns. Details of the manufacturing process are set out in the seriesof steps described below:

A solution comprising a bioactive agent such as, but not limited to, animmunogen or immunogenic substance(s) is dissolved into the slurrydescribed above prior to cross-linking. The resulting composition isthen allowed to fall in drops, or in a continuous stream, into across-linking solution of 1-10% sodium triphosphate solution.Alternatively, the slurry can be spray-atomized into an aqueous solutioncontaining 1-10% sodium triphosphate. A chitosan/tripolyphosphatemolecular mass ratio of at least about 4:1 is maintained. After ahardening period of 30-180 minutes, the wet particles beads or threadsare harvested from the cross-linking bath by any suitable means wellknown in the art (e.g., screening, filtration, centrifugation, and thelike) and soaked in a saturated sugar solution followed by mixing in anyfood acceptable desiccation compounds such as silica gel, starchgranules and the like for further dewatering. The sodium triphosphateand the sugar saturated solutions can be reused for cross-linking morebatches of chitosan slurry. The silica gel can also be washed,sterilized, and reused to dewater additional batches. The semi-dryparticles are mixed in the feed formula for subsequent pelleting orextrusion. Alternatively, the semi-dry particles can be further driedusing conventional processes well known in the art such as, but notlimited to, freeze drying, vacuum drying, fluidized bed drying andtunnel drying. The dried material is then milled and sieved to theappropriate particle size class if necessary. The final sized particlescan be mixed directly with the feed materials for subsequent pelletingor extrusion, or it can be mixed with edible oil for top-coating of astandard commercially available feed for oral administration.

FIG. 1 demonstrates the process equipment which includes an airtight,stainless steel steam jacketed insulated mixing vessel (A) provided witha high-speed homogenizer from 250-20,000 RPM. The temperature invessel-A is controlled from about 20° C. to 80° C. The mucoadhesiveslurry containing the bioactive agent is prepared in vessel-A asdescribed above. Nitrogen gas pressure from about 5-25 psi is thenapplied and controlled by Pressure regulator-1 to force the slurrythrough Feed line 2 (FIG. 1). The process tank (Vessel-B) is where themucoadhesive polymer hydrogel beads or threads are formed. The airtightall stainless steel vessel is equipped with a liquid jet unit connectedto Feed line-2, an exhaust system (Exhaust filter-3), and a removablestainless mesh basket (Mesh basket-4) that is raised about 10 cm fromthe vessel bottom. The liquid height in vessel-B is sufficient tomaintain a minimum of 80 cm distance between the jet and the surface ofthe cross-linking solution in the tank which is necessary for allowingthe stream of beads or continuous threads to settle at the bottom whilehardening, thereby clearing the solution surface to accept freshlyintroduced material. In one embodiment of the present invention, thedensity of the slurry can be further adjusted by the addition ofinsoluble salts such as, but not limited, to calcium carbonate orcalcium sulfate. This will allow for a rapid sinking of the beads orcontinuous threads to the tank bottom, thereby clearing the solutionsurface for freshly introduced material. Otherwise material mayaccumulate at the surface and prevent effective cross-linking of freshlyintroduced particles or threads. The hydrogel particles or threads aretrapped within the mesh basket in vessel-B and are allowed to harden for30-180 min. The Mesh basket-4 is then raised above the surface of thecross-linking solution and the beads or threads are allowed to drip dryfor 30-120 min before being transferred to a third stainless steelvessel (Vessel-C) for further dewatering. FIG. 2 shows hydrogel stringsbefore (upper picture) and after (bottom picture) harvesting from vesselB.

Vessel-C is similar to Vessel-B and serves for sugar soaking anddewatering of the hydrogel particles. Initially the tank filled with asolution containing at least 40% sugars or, more preferably, a sugarsaturated solution. The hydrogel particles or threads are soaked in thesugar solution under vacuum of from about 1-100 mBARS for 30-60 min thenallowed to drip dry by raising the mesh basket as described above. Thesugar solution is removed from the tank and a thick layer of foodcompatible desiccating agent such as, but not limited to, silica gelbeads or starch granules, is introduced to vessel-B. The mesh basketcontaining the hydrogel particles or threads is placed on top of thedesiccating agent layer in vessel-C. A vacuum pressure from about 1-100mBARS is applied again for additional 120 min. The semi-dry materialcontaining 10-20% moisture is then removed from the vessel and choppedto smaller size and directly incorporated in a feed formula forpelleting or extrusion. Alternatively, the semi-dry material can befurther dried to a moisture content of below about 10%, followed bymilling and sieving through mesh screens of an appropriate size (e.g.,from about 50-500 micron). The sodium triphosphate solution in Vessel-Band sugar saturated solution in Vessel-C can be reused for cross-linkingmore batches of chitosan slurry, and the silica gel in Vessel-C washed,sterilized, and reused to dewater additional batches.

The diameter of beads or strings produced by this method will varydepending on the jet diameter and stream velocity. The jet diameterfound to be useful in producing the particles was in the range from 50to 7000 microns. The vertical jet velocity found to be useful was in therange of 0.1 cm³/sec to 10 cm³/sec. One of the advantages of the presentinvention is in the controlling of jet velocity by the nitrogen gaspressure applied in Vessel-A and the simple sterilization procedurewhich effectively eliminates the cumbersome use of a typical pumpingsystem.

Additionally, in an alternative embodiment, the particles are removedfrom the mesh basket and directly placed in a drying unit without thedewatering step in Vessel-C. Due to the sugars saturation step, thehydrogel particles contain only about 30-40% water and thus require aminimal drying process to further reduce the moisture content to below10%.

In another embodiment, the slurry is transferred from Vessel-A under apressure of nitrogen gas to a separate pressure vessel connected to Feedline-2, and Vessel-C is used for sugar saturation while a separatesimilar Vessel-D is used for desiccation. This allows for continuousoperation of the system, whereby additional chitosan/bioactive agentslurry is produced while Vessels-B, C and D are occupied with theformation and dewatering of the hydrogel particles or strings.

EXAMPLES Example 1

Production of mucoadhesive polymer particles

In a 400 L airtight steam jacketed stainless steel vessel, 180 L ofsterile distilled water is added and warmed to 50° C. Three L of glacialacetic acid is carefully added (with mixing) to 17 liters of distilledwater in an open flame hood. The diluted acetic acid solution is thenslowly added to the distilled water in the steam jacketed stainlesssteel tank. Chitosan (4 kg high viscosity chitosan, Sigma, St. Louis,Mo.) is slowly added to the warmed acetic acid solution under a vigoroushomogenizing (10,000 RPM) until completely dissolved. The chitosansolution is cooled to room temperature and the pH adjusted to 6.2 with50% sodium hydroxide solution. Liquid soy lecithin (6 kg,Archer-Daniels-Midland Co., Decatur, Ill.) is added to the chitosansolution and the solution emulsified for 15 minutes under vigoroushomogenization (10,000 RPM).

A solution of bacterin containing 5 million injectable doses is added tothe chitosan slurry mixture prepared above. A pressure of 15 psi is thenapplied to the vessel and the slurry forced through ¼ quarter of inchfeed line connected to a liquid jet head unit located at the center topcover of a 1000 L airtight steam jacketed stainless steel flat bottomvessel. The vessel is equipped with #10-12-mesh stainless steel basketlocated about 10 cm above the vessel bottom. The vessel contains 300 Lof 10% w/w sodium triphosphate solution. The chitosan slurry is forcedthrough a 5 mm liquid jet head unit that produces a uniform stream ofthe slurry into the sodium triphosphate solution. Uniform size stringsof hydrogel are instantly formed and sink to the bottom of the meshedbasket. The hydrogel strings are allowed to harden for 2 hours in thesodium triphosphate solution. After fully hardened, the meshed basket israised above the solution surface and allowed to drip for an additionalhour.

The mesh basket containing the hydrogel string material is thentransferred to a similar 1000 L airtight stainless steel flat bottomvessel containing about 100 liter of sucrose saturated solution and avacuum of 10 mBARS is applied for 30 minutes facilitating the absorptionof the sugars by the hydrogel particles. The mesh basket is then raisedabove solution surface and the material is allowed to drip dry for 2hours. The mesh basket containing the sugar saturated hydrogel materialis then transferred to another similar 1000 litter airtight stainlesssteel flat bottom vessel containing about 100 kg of #35-60 mesh foodgrade and sterile silica gel. The vacuum on the tank is reduced to about10 mBARS for 1 hour to provide further dewatering. The semi-dry hydrogelmaterial containing about 20% moisture is chopped to small pieces ofless than 1 mm in size for incorporation in a feed formula for pelletingor extrusion. Alternatively, the semi-dry hydrogel material can befurther dehydrated to below 10% moisture in a vacuum drier or fluidizedbed drier. Once the material attains a moisture content of less than10%, it can be milled and sieved to provide final material of less than100 micron particle size for incorporation in feed formula for pelletingor extrusion. This final sieved material can also be mixed in edible oiland used to top coat ready-made feed pellets.

Example 2

Production of Mucoadhesive Polymer Particles using high fructose cornsyrup (HFCS).

Hydrogel Particles or strings are prepared as in Example 1. Sterile HFCSsolution (Archer-Daniels-Midland Co., Decatur, Ill.) containing 55%fructose is used to saturate the hydrogel particles. 100 L of HFCSsolution is added to a 1000 L airtight stainless steel flat bottomvessel and hydrogel particles are allowed to soak in the HFCS for about30 minutes as described above in example 1. The particles are furtherdewatered in silica gel and semi-dry hydrogel material containing 20%moisture is obtained following the dehydration procedure described inexample 1. The semi-dry hydrogel material is further dried in afluidized bed drier at 50° C. to achieve a moisture level less than 10%.The resulting dried material is then milled using an industrial hammermill and the powder is sieved to less than 100-microm-particle size.

Example 3

Production of Mucoadhesive Polymer Particles containing SalmonidRickettsial Septicaemia (SRS) Vaccine.

The mucoadhesive polymer slurry at pH 6.2 was prepared as described inExample 1. A solution containing attenuated SRS vaccine (5×10¹⁷/ml SRSbacterin) (commercially available from Centrovet, Santiago, Chile) wasmixed with yeast extract immunostimulator (500 g beta glucan, AHDInternational, Atlanta, Ga.) and added into the slurry. The slurry wasthen injected into 10% w/v sodium triphosphate solution. The hydrogelmaterial was allowed to harden for 2 hour and then saturated withsucrose. It was then further dewatered in silica gel, freeze dried, andmilled to particle size lower than 100 micron.

An Enzyme-Linked ImmunoSorbant Assay (ELISA) was used to validate thepresence of intact SRS vaccine in the mucoadhesive polymer particles.The encapsulated SRS vaccine produced above was homogenized inPhosphate-Buffered Saline (PBS) at different pH levels. FIG. 3 shows theoptimum condition for immunogen release from the particles. This wasfollowed by enzymatic treatment at 37° C. with continuoushomogenization. The supernatant from the digested antigen preparationwas used for ELISA and real-time Polymerase Chain Reaction (PCR) assay.Particles were homogenized and extracted in 10 mM PBS pH 6.3 containingchitosanase enzyme. Total protein was quantified using the Bradfordmicrotiter assay (Bradford 1976). ELISA's were typical sandwich style inwhich the microtiter plates were coated with primary sheep antibody P.salmonis (Sigma). The SRS antibodies were captured overnight at 4° C.,and the plate was reacted with a secondary anti-sheep IgG (Sigma HRPconjugate), followed by the anti-sheep alkaline phosphatase conjugate(Jackson Immunoresearch, West Grove, Pa.). The alkaline phosphatase isdetected with para-nitrophenyl phosphate and read at 405 nm on aSpectroMax plate reader (Molecular Devices, Sunnyvale, Calif.).

Example 4

Production of Bioadhesive particles with enhanced immunogenicproperties.

In a 400 L airtight steam jacketed stainless steel vessel, 180 L ofsterile distilled water is added and warmed to 50° C. Three L of glacialacetic acid is carefully added (with mixing) to 17 liters of distilledwater in an open flame hood. The diluted acetic acid solution is thenslowly added to the distilled water in the steam jacketed stainlesssteel tank. Chitosan (4 kg high viscosity chitosan, Sigma, St. Louis,Mo.) is slowly added to the warmed acetic acid solution under a vigoroushomogenizing (10,000 RPM) until completely dissolved. The chitosansolution is cooled to room temperature and the pH adjusted to 6.2 with50% sodium hydroxide solution. Instant Inulin (60 kg, Cargil,Minneapolis, Minn.) and liquid soy lecithin (6 kg,Archer-Daniels-Midland Co., Decatur, Ill.) are added to the chitosansolution and the solution emulsified for 15 minutes under vigoroushomogenization (10,000 RPM). A solution containing 10 ml (equivalent to5 million injectable doses) of attenuated bacterin vaccine, viralvaccine or yeast lysate containing proteins recombinants assembly in aVLP platform (available commercially from Novartis animal Health,Greensboro, N.C. USA.) are mixed with yeast extract immunostimulator(500 g beta glucan, AHD International, Atlanta, Ga.) and added into thechitosan slurry. The slurry is then injected into 10% w/v sodiumtriphosphate solution. The hydrogel material is allowed to harden for 2hour and then saturated with sucrose. The hydrogel is further dewateredin Sharples centrifuge and dried in a freeze dryer. The resulting driedmaterial is then milled using an industrial hammer mill and the powderis sieved to less than 100-microm-particle size.

Example 5

Production of Bioadhesive particles containing a viral antigen vaccineagainst viral infections in fish.

The composition of the present invention is also effective in oralvaccination against viral infections such as infectious salmon anemiavirus (ISAV), infectious pancreatic necrosis virus (IPNV), salmonswimbladder sarcoma virus (SSSV), etc. One injectable dose may beequivalent to 2-20 micro liter solution of attenuated virus vaccine oryeast lysate containing recombinant proteins obtained from sequences ofisolated virus. Both types of vaccines are available commercially fromCentrovet, Santiago, Chile or Novartis animal Health, Greensboro, N.C.USA. A typical formulation involves the preparation of slurry asdescribed in Example 1. A solution containing 5 million doses ofattenuated virus or proteins recombinants are mixed with yeast extractimmunostimulator (500 g beta glucan, AHD International, Atlanta, Ga.)and added into the slurry. The slurry is then injected into 10% w/vsodium triphosphate solution. The hydrogel material is allowed to hardenfor 2 hour and then saturated with sucrose. It is then further dewateredin silica gel or Sharples centrifuge, freeze dried, vacuum dried orfluidized bed dried, and milled to particle size lower than 100 micron.

Example 6

Production of Bioadhesive particles containing a viral antigen vaccineagainst ISAV.

A 10 ml of yeast lysate solution (equivalent to 5 million injectabledoses) containing proteins recombinants obtained from sequences ofisolated virus of a Chilean origin (available commercially fromCentrovet, Santiago, Chile) is mixed with yeast extract immunostimulator(500 g beta glucan, AHD International, Atlanta, Ga.) and added into aslurry as described in example 1. The slurry is then injected into 10%w/v sodium triphosphate solution. The hydrogel material is allowed toharden for 2 hour and then saturated with sucrose. The hydrogel isfurther dewatered in Sharples centrifuge and dried in a freeze dryer.The dried material is milled to particle size lower than 100 micron.

Example 7

Production of Bioadhesive particles containing a viral antigen vaccineagainst IPNV.

The economical loss due to IPN is significant in the salmon farmingindustry, and outbreaks may occur both in fresh water pre-smolt andpost-smolt salmon after transferred to sea-water. An IPNV infection maypersist without any signs of disease, but it may reactivate with newoutbreaks in post-smolts after transfer to seawater. Effectiveinjectable vaccines are available, but difficult to apply in small earlypre-smolt salmon juveniles. An oral application using virus-likeparticle (VLP) as a platform and the formulation of the currentinvention is produced. A 10 ml of yeast lysate solution (equivalent to 5million injectable doses) containing proteins recombinants assembly in aVLP platform (available commercially from Novartis animal Health,Greensboro, N.C. USA.) is mixed with yeast extract immunostimulator (500g beta glucan, AHD International, Atlanta, Ga.) and added into a slurryas described in example 1. The slurry is then injected into 10% w/vsodium triphosphate solution. The hydrogel material is allowed to hardenfor 2 hour and then saturated with sucrose. The hydrogel is furtherdewatered in Sharples centrifuge and dried in a freeze dryer. Theresulting dried material is then milled using an industrial hammer milland the powder is sieved to less than 100-microm-particle size.

Example 8

Production of Storage Stable Bioadhesive particles containing a viralantigen vaccine against viral infections in fish.

The final drying step of the composition of the present invention may becarried out in a way that allowed the formation of a sugar glass matrixsurrounded the vaccine particles. Such a glassy formation stabilizes andprotects the vaccine under unfavorable storage conditions of hightemperature and humidity. Oral bacterin vaccines against a bacterialdisease such as an SRS or viral vaccines against virus infections suchas ISAV or IPNV were prepared as described in Example 6 or 7 and thefinal drying step of the sugar loaded thin threads or strings were driedin such a way that a sugar glass is formed. The thin threads were loadedon a 13×10″ tray (13×10 inch) at a loading capacity of 800 g/sq ft andplaced in a freeze drier (Virtis Advantage, Virtis, Gardiner, N.Y.). TheCondenser is chilled to—50° C., shelf temperature was adjusted to 40°degree C. and the material allowed to warm up to about 35° C. (measuredby a pair of temperature sensors plugged in the wet material). Vacuumwas then initiated and controlled at about 2500 mTORR through anexternal vacuum controller, (Thyr-Cont, Electronic, GmbH). As vacuumpulled down the product temperature fall and stabilized at about ±2° C.After 16 hours, the product temperature had increased to about +10° C.At this point, a full vacuum pressure was applied and shelf temperaturerose to 50° C. Twelve hours after establishing full vacuum pressure, thedried product was taken out of the freeze drier and milled to particlesize lower than 100 micron.

Example 9

Production of Atlantic salmon feed containing SRS immunogenicmicroparticles

Fifteen kg of dry powder of SRS immunogenic microparticles was preparedas in Example 3 and mixed with 30 kg of fish oil. The oily mixture wassprayed on 1000 kg of standard commercial feed for Atlantic salmonjuveniles (Ewos, Km 20 Coronel, Concepción, Chile) and the oralvaccination feed was stored in 4° C. during its use.

Example 10

Production of mucoadhesive polymer particles containing Mycoplasmahyopneumoniae (M. hyo) vaccine.

Mycoplasma hyopneumoniae (M. hyo) is a widespread pathogen, found inswine herds throughout the world. The organism is associated with thedevelopment of bronchopneumonia. The disease leads to severeproductivity losses by way of reduced weight gain and poor foodconversion rates in the growing pigs. To orally vaccinate the pig, theslurry (pH 6.2) was prepared as described in Example 2. A solutioncontaining 5 million doses of attenuated M. hyo vaccine commerciallyavailable from Boehringer Ingelheim Limited, Berkshire UK. is mixed with500 g yeast extract immunostimulator (pure beta glucan, AHDInternational, Atlanta, Ga.) and added into the slurry. The slurry isthen injected into 10% w/v sodium triphosphate solution containing HFCS.The hydrogel material is allowed to harden for 2 hour and then furtherdewatered and chopped to a particle size less than 1 mm and keptrefrigerated until incorporated in feed formula for pellet extrusion.

Example 11

Oral vaccination of pigs against M. hyo using the immunogenicmicroparticles of the present invention.

Semi-dry immunogenic particles produced as described in example 10 areincorporated in standard feed formula for growing pigs and a pelletedfeed is produced. Growing pigs are fed once a day with a diet containing1 dose of the injectable vaccine for a total of 5 days. Growthperformances of vaccinated and non-vaccinated pigs are recorded.

Example 12

Nasal vaccination of pigs against M. hyo using the immunogenicmicroparticles of the present invention.

The semi-dry hydrogel particles produced as described in examples 2 and10 are dried in a vacuum drier to further reduce the moisture content tobelow 10%. The dry material is milled using an industrial hammer milland sieved to particle size less than 30 micron. Growing pigs areimmunized with the immunogenic microparticles via the respiratory tractand growth performances of vaccinated and non-vaccinated pigs recorded.

References

The contents of all references cited herein are hereby incorporated byreference herein for all purposes.

Aral, C. and Akbuga, A, J. (1998). Alternative approach to thepreparation of chitosan beads. International of Journal ofPharmaceutics, 168:9-15.

Bradford, M. M. (1976). A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principalof protein-dye binding. Anal. Biochem. 72:248-254.

Bodmeier, R., Oh, K. H. and Pramar, Y. (1989). Preparation andevaluation of drug containing chitosan beads. Drug Development andIndustrial Pharmacy, 15,1475-1494.

Calvo, P., Remun, A., N-Lopez, C., Vila-Jato, J. L. and Alonso, M. J.(1997). Novel hydrophilic chitosan-polyethylene oxide nanoparticles asprotein carriers. Journal of Applied Polymer Science, 63: 125-132.

Chopra, S., S. Mandi, et al. (2006). “Advances and potentialapplications of chitosan derivatives as mucoadhesive biomaterials inmodern drug delivery.” J. Pharm. Pharmacol. 58(8): 1021-1032.

Dang, J. M. and K. W. Leong (2006). “Natural polymers for gene deliveryand tissue engineering.” Adv. Drug Deliv. Rev. 58(4): 487-499.

Davis, S. S. (2006). “The use of soluble polymers and polymermicroparticles to provide improved vaccine responses after parenteraland mucosal delivery.” Vaccine 24(2): 7-10.

Huang, Y. C., Chiang, C. H. and Yeh, M. K. (2003), OptimizingFormulation Factors in Preparing Chitosan Microparticles by Spray-dryingMethod, Journal of Microencapsulation, 20(2: 247-260.

Kang, M. L., H. L. Jiang, et al. (2007). “Pluronic F127 enhances theeffect as an adjuvant of chitosan microspheres in the intranasaldelivery of Bordetella bronchiseptica antigens containingdermonecrotoxin.” Vaccine 25(23): 4602-4610.

Kim, T. J., K. H. Kim, et al. (2007). “Stimulation of mucosal andsystemic antibody responses against recombinant transferrin-bindingpro{acute over (t)}ein B of Actinobacillus pleuropneumoniae withchitosan after tracheal administration in piglets.” J. Vet. Med. Sci.69(5): 535-539.

Malik, D. K., S. Baboota, et al. (2007). “Recent advances in protein andpeptide drug delivery systems. .” Curr. Drug Deliv. 4(2): 141-151.

Rege P. R, Gramise R. J, Block L. H. (2003). Spray-dried chitinosans.Part-I: preparation and characterization. Int J Pharm. 252:41-51.

Shiraishi, S., Imai, T. and Otagiri, M. (1993). Controlled release ofindomethacin by chitosan-polyelectrolyte complex: optimization and invivo/in vitro evaluation. Journal of Controlled Release, 25: 217-225.

Shu, X. Z. and Zhu, K. J., (2000). A novel approach to preparetripolyphosphate/chitosan complex beads for controlled drug delivery.International of Journal of Pharmaceutics, 201,51-58.

van der Lubben, I. M., J. C. Verhoef, et al. (2001). “Chitosan formucosal vaccination.” Advanced Drug Delivery Reviews 52 (2): 139-144.

van der Lubben, I. M., J. C. Verhoef, et al. (2001). “Chitosanmicroparticles for oral vaccination: preparation, characterization andpreliminary in vivo uptake studies in murine Peyer's patches.”Biomaterials 22(7): 687-694.

That which is claimed is:
 1. An immunogenic substance for oral or nasalvaccination comprising a vaccine, a cross-linked mucoadhesive polymer,10-60 wt % of one or more sugars, 3-10 wt % of one or moreoligosaccharides and 1-10 wt % of one or more electrolytes, wherein theimmunogenic substance is prepared by a method comprising: (a) dissolvingthe mucoadhesive polymer and the one or more oligosaccharides in anaqueous solution; (b) mixing the vaccine with the resulting solutionfrom step (a) to form a slurry; (c) cross-linking the mucoadhesivepolymer in the slurry with the one or more electrolytes to form hydrogelparticles; (d) soaking the hydrogel particles in a solution saturatedwith the one or more sugars to make semi-dry particles; and (e) dryingthe semi-dry particles to form the immunogenic substance.
 2. Theimmunogenic substance of claim 1, wherein the vaccine is a bacterinvaccine, a viral vaccine, or a recombinant vector that expresses a geneencoding an immunogenic protein.
 3. The immunogenic substance of claim1, wherein the vaccine is selected from the group consisting of (a)recombinant vectors comprising a gene encoding an immunogenic protein,(b) intact inactive, attenuated or infectious viral particles, (c)intact killed, attenuated or infectious prokaryotes, (d) intact killed,attenuated or infectious protozoans, and (e) intact killed, attenuatedor infectious multicellular pathogens.
 4. The immunogenic substance ofclaim 1, wherein the vaccine is against infectious salmon anemia virus(ISAV), Salmonid Rickettsial Septicaemia (SRS) or infectious pancreaticnecrosis virus (IPNV).
 5. The immunogenic substance of claim 1, whereinthe mucoadhesive polymer is a polysaccharide.
 6. The immunogenicsubstance of claim 5, wherein the polysaccharide is selected from thegroup consisting of chitosan, hyaluronic acid, alginate, cationic guar,and derivatives thereof.
 7. The immunogenic substance of claim 1,wherein the sugar is a monosaccharide or a disaccharide.
 8. Theimmunogenic substance of claim 7, wherein the disaccharide is selectedfrom the group consisting of sucrose and trehalose.
 9. The immunogenicsubstance of claim 1, wherein the one or more electrolytes are one ormore divalent or polyvalent cations or anions.
 10. The immunogenicsubstance of claim 1, wherein the one or more electrolytes are one ormore salts of Ca, Zn, Al, triphosphate or hexametaphosphate.
 11. Theimmunogenic substance of claim 1, wherein the immunogenic substanceconsists of the vaccine, the cross-linked mucoadhesive polymer, the oneor more sugars, the one or more oligosaccharides and the one or moreelectrolytes, wherein the one or more sugars are selected from the groupconsisting of fructose, sucrose, dextrose, and trehalose, wherein theone or more oligosaccharides are selected from the group consisting ofinulin and fructooligosaccharides (FOS), and wherein the mucoadhesivepolymer is a polymer that is not a dimer.
 12. The immunogenic substanceof claim 1, wherein the immunogenic substance consists of the vaccine,the cross-linked mucoadhesive polymer, the one or more sugars, the oneor more oligosaccharides, the one or more electrolytes, and anadditional agent, wherein the one or more sugars are selected from thegroup consisting of fructose, sucrose, dextrose, and trehalose, whereinthe one or more oligosaccharides are selected from the group consistingof inulin and fructooligosaccharides (FOS), and wherein the mucoadhesivepolymer is a polymer that is not a dimer, and wherein the additionalagent is selected from the group consisting of a phospholipid, betaglucan and a combination thereof.
 13. An animal feed comprising theimmunogenic substance of claim
 1. 14. A method for vaccination of ananimal, comprising administering the immunogenic substance of claim 1 tothe animal.
 15. The method of claim 14, wherein the animal is a fish,salmonid, bird or a mammal.
 16. The method of claim 14, wherein thevaccine is a bacterin vaccine, a viral vaccine, or a recombinant vectorthat expresses a gene encoding an immunogenic protein.
 17. The method ofclaim 14, wherein the vaccine is against infectious salmon anemia virus(ISAV), Salmonid Rickettsial Septicaemia (SRS) or infectious pancreaticnecrosis virus (IPNV).
 18. The method of claim 14, wherein theimmunogenic substance is spray coated on a feed pellet.
 19. The methodof claim 14, wherein the immunogenic substance consists of the vaccine,the cross-linked mucoadhesive polymer, the one or more sugars, the oneor more oligosaccharides and the one or more electrolytes, wherein theone or more sugars are selected from the group consisting of fructose,sucrose, dextrose, and trehalose, wherein the one or moreoligosaccharides are selected from the group consisting of inulin andfructooligosaccharides (FOS), and wherein the mucoadhesive polymer is apolymer that is not a dimer.
 20. The method of claim 14, wherein theimmunogenic substance consists of the vaccine, the cross-linkedmucoadhesive polymer, the one or more sugars, the one or moreoligosaccharides, the one or more electrolytes, and an additional agent,wherein the one or more sugars are selected from the group consisting offructose, sucrose, dextrose, and trehalose, wherein the one or moreoligosaccharides are selected from the group consisting of inulin andfructooligosaccharides (FOS), and wherein the mucoadhesive polymer is apolymer that is not a dimer, and wherein the additional agent isselected from the group consisting of a phospholipid, beta glucan and acombination thereof.